Neisseria meningitidis serogroup B Glycoconjugates

ABSTRACT

The present invention pertains generally to  Neisseria meningitidis  serogroup B glycoconjugates. More particularly, the invention pertains to glycoconjugates formed from a  Neisseria meningitidis  serogroup B capsular oligosaccharide derivative (MenB OS derivative) in which sialic acid residue N-acetyl groups are replaced with N-acyl groups. The invention also pertains to vaccine formulations containing the glycoconjugates, methods of making the vaccine formulations, and methods of using the vaccine formulations to treat or prevent  Neisseria meningitidis  serogroup B or  E. coli  K1 disease in a mammalian subject.

CROSS-REFERENCE TO RELATED APPLICATION

This application is related to provisional patent application Ser. No.60/024,454, filed Aug. 27, 1996, from which priority is claimed under 35USC §119(e)(1) and which is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention pertains generally to novel Neisseria meningitidisserogroup B glycoconjugates. More particularly, the invention pertainsto glycoconjugates formed from a Neisseria meningitidis serogroup Bcapsular oligosaccharide derivative (MenB OS derivative) in which sialicacid residue N-acetyl groups have been replace with N-acyl groups, andmethods of making and using those glycoconjugates.

2. Background of the Invention

Neisseria meningitidis is a causative agent of bacterial meningitis andsepsis. Meningococci are divided into serological groups based on theimmunological characteristics of capsular and cell wall antigens.Currently recognized serogroups include A, B, C, D, W-135, X, Y, Z and29E. The polysaccharides responsible for the serogroup specificity havebeen purified from several of these groups, including A, B, C, D, W-135and Y.

N. meningitidis serogroup B (“MenB”) accounts for approximately 50percent of bacterial meningitis in infants and children residing in theU.S. and Europe. The organism also causes fatal sepsis in young adults.In adolescents, experimental MenB vaccines consisting of outer membraneprotein (OMP) vesicles have been found to be approximately 50%protective. However, no protection has been observed in vaccinatedinfants and children, the age groups at greatest risk of disease.Additionally, OMP vaccines are serotype- and subtype-specific, and thedominant MenB strains are subject to both geographic and temporalvariation, limiting the usefulness of such vaccines.

Effective capsular polysaccharide-based vaccines have been developedagainst meningococcal disease caused by serogroups A, C, Y and W135.However, similar attempts to develop a MenB polysaccharide vaccine havefailed due to the poor immunogenicity of the capsular MenBpolysaccharide (termed “MenB PS” herein). MenB PS is a homopolymer of(N-acetyl (α2→8) neuraminic acid. Escherichia coli K1 has the identicalcapsular polysaccharide. Antibodies elicited by MenB PS cross-react withhost polysialic acid (PSA). PSA is abundantly expressed in fetal andnewborn tissue, especially on neural cell adhesion molecules (“NCAMs”)found in brain tissue. PSA is also found to a lesser extent in adulttissues including in kidney, heart and the olfactory nerve. Thus, mostanti-MenB PS antibodies are also autoantibodies. Such antibodiestherefore have the potential to adversely affect fetal development, orto lead to autoimmune disease.

MenB PS derivatives have been prepared in an attempt to circumvent thepoor immunogenicity of MenB PS. For example, C₄-C₈ N-acyl-substitutedMenB PS derivatives have been described. See, EP Publication No. 504,202B, to Jennings et al. Similarly, U.S. Pat. No. 4,727,136 to Jennings etal. describes an N-propionylated MenB PS molecule, termed “NPr-MenB PS”herein. Mice immunized with NPr-MenB PS glycoconjugates were reported toelicit high titers of IgG antibodies. Jennings et al. (1986) J. Immunol.137:1708. In rabbits, two distinct populations of antibodies,purportedly associated with two different epitopes, one shared by nativeMenB PS and one unshared, were produced using the derivative.Bactericidal activity was found in the antibody population that did notcross react with MenB PS. Jennings et al. (1987) J. Exp. Med. 165:1207.The identity of the bacterial surface epitope(s) reacting with theprotective antibodies elicited by this conjugate remains unknown.

Although the above-described MenB PS derivatives are capable ofeliciting a significant anti-MenB PS response, responding antibodiesstill include a significant proportion of molecules that arecross-reactive with polysialic acid residues in host tissue, andtherefore autoreactive. Thus, to date, no approach which has been takenwith respect to MenB vaccine development has been successful inproviding a safe and effective vaccine against MenB. Accordingly, thereremains a need to provide MenB immunogens which can be used in vaccineformulations, wherein the immunogens do not elicit the production ofantibodies in immunized animals that are cross-reactive with host tissueand can be thus used in the prevention or treatment of MenB disease.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that a substantiallyhomogenous preparation of MenB oligosaccharide (MenB OS) derivativefragments, and glycoconjugates made from those fragments, provide highlyeffective immunogens for use in anti-MenB vaccine preparations.Antibodies elicited in immunized animals by these MenB OS derivativefragments do not substantially cross-react with host tissue asdetermined using several binding assays described herein, and aretherefore not autoreactive. Since the present MenB OS fragments do notelicit the formation of autoreactive molecules, they provide a safe andefficacious vaccine component for use in the prevention of MenB and E.coli K1 disease.

Accordingly, in one embodiment, the subject invention is directed to aglycoconjugate comprising a MenB OS derivative having sialic acidresidue N-acetyl groups replaced with N-acyl groups, wherein the MenB OSderivative is covalently attached to a carrier molecule and has anaverage degree of polymerization (Dp) of about 10 to about 20.

In another embodiment, the subject invention is directed to aglycoconjugate comprising a MenB OS derivative having sialic acidresidue N-acetyl groups replaced with N-propionyl groups, wherein theMenB OS derivative is covalently attached to a tetanus toxoid proteincarrier and has an average Dp of about 12 to about 18.

In yet another embodiment, the invention is directed to a method forproducing a glycoconjugate comprising:

(a) providing a heterogenous population of MenB OS derivatives whereinsialic acid residue N-acetyl groups have been replaced with N-acylgroups;

(b) obtaining a substantially homogenous group of MenB OS derivativesfrom the population of (a) wherein the MenB OS derivatives have anaverage Dp of about 10 to 20;

(c) introducing a reactive group at a nonreducing end of the derivativesobtained in step (b) to provide single end-activated MenB OSderivatives; and

(d) covalently attaching the end-activated MenB OS derivatives to acarrier molecule to provide a MenB OS glycoconjugate comprisingsubstantially homogenous sized MenB OS moieties.

In still a further embodiment, the invention is directed to a method forproducing a glycoconjugate comprising:

(a) providing a heterogenous population of MenB OS derivatives whereinsialic acid residue N-acetyl groups have been replaced with N-propionylgroups;

(b) obtaining a substantially homogenous group of MenB OS derivativesfrom the population of (a) wherein the MenB OS derivatives have anaverage Dp of about 12 to 18;

(c) introducing a reactive group at a nonreducing end of the derivativesobtained in step (b) to provide single end-activated MenB OSderivatives; and

(d) covalently attaching the end-activated MenB OS derivatives to atetanus toxoid carrier molecule to provide a MenB OS/tetanus toxoidglycoconjugate comprising substantially homogenous sized MenB OSmoieties.

In still further embodiments, the subject invention relates toglycoconjugates produced by these methods, to vaccine compositionscomprising the glycoconjugates in combination with a pharmaceuticallyacceptable excipient, and to methods of forming the vaccinecompositions.

In another embodiment, the subject invention is directed to a method forpreventing or treating MenB and/or E. coli K1 disease in a mammaliansubject comprising administering a therapeutically effective amount ofthe above vaccine compositions to the subject.

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the CONJ-1 NPr-MenB OS derivative-based glycoconjugateproduced in the practice of the invention.

FIG. 2 depicts the CONJ-2 NPr-MenB OS derivative-based glycoconjugateproduced in the practice of the invention.

FIG. 3 depicts the CONJ-3 NPr-MenB OS derivative-based glycoconjugateproduced in the practice of the invention.

FIG. 4 depicts the CONJ-4 NPr-MenB OS derivative-based glycoconjugateproduced in the practice of the invention.

FIGS. 5A and 5B depict chromatograms taken during preparation of acontrol NPr-MenB PS//CRM₁₉₇ glycoconjugate before (FIG. 5A) and after(FIG. 5B) covalent attachment of the saccharides to the protein carrieras described in Example 2.

FIG. 6 depicts the results of the ELISA described in Example 3evaluating the production of an IgG anti-NPr-MenB PS antibody responsein animals immunized with a vaccine composition containing the controlglycoconjugates.

FIG. 7 depicts the results of the ELISA described in Example 4evaluating the IgG subclass of the antibody response elicited by acontrol glycoconjugate vaccine composition.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of immunology, microbiology, molecularbiology and recombinant DNA techniques within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., Sambrook,et al. Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); DNACloning: A Practical Approach, vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic AcidHybridization (B. Hames & S. Higgins, eds., 1985); Transcription andTranslation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R.Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning(1984); and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weirand C. C. Blackwell eds., 1986, Blackwell Scientific Publications).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

I. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

As used herein, a “MenB PS derivative” refers to a molecule obtained bythe chemical modification of the native capsular polysaccharide of MenB.Such MenB PS derivatives include, but are not limited to, MenB PSmolecules which have been modified by the substitution of sialic acidresidue N-acetyl groups of the native molecule with appropriate acylgroups, such as C₃-C₈, and higher, acyl groups wherein the term “acylgroup” encompasses any acylated linear, branched, aliphatic or aromaticmolecule. A particularly preferred MenB PS derivative for use hereincomprises the substitution of N-propionyl groups for N-acetyl groups ofnative MenB PS (termed “NPr-MenB PS” herein). Methods for synthesizingN-acyl-substituted MenB PS derivatives, including NPr-MenB PS, are knownin the art and described in e.g., U.S. Pat. No. 4,727,136 to Jennings etal. and EP Publication No. 504,202 B, also to Jennings et al.

An “antigen” is defined herein to include any substance that may bespecifically bound by an antibody molecule. An “immunogen” is an antigenthat is capable of initiating lymphocyte activation resulting in anantigen-specific immune response. Such activation generally results inthe development of a secretory, cellular and/or antibody-mediated immuneresponse against the immunogen. Usually, such a response includes but isnot limited to one or more of the following effects; the production ofantibodies from any of the immunological classes, such as IgA, IgD, IgE,IgG or IgM; the proliferation of B and T lymphocytes; the provision ofactivation, growth and differentiation signals to immunological cells;expansion of helper T cell, suppressor T cell, and/or cytotoxic T celland/or γδ T cell populations. Immunogens therefore include any moleculewhich contain one or more antigenic determinants (e.g., epitopes) thatwill stimulate a host's immune system to initiate such anantigen-specific response.

By “epitope” is meant a site on an antigen to which specific B cells andT cells respond. The term is also used interchangeably with “antigenicdeterminant” or “antigenic determinant site.” A peptide epitope cancomprise 3 or more amino acids in a spatial conformation unique to theepitope. Generally, an epitope consists of at least 5 such amino acidsand, more usually, consists of at least 8-10 such amino acids. Methodsof determining spatial conformation of amino acids are known in the artand include, for example, x-ray crystallography and 2-dimensionalnuclear magnetic resonance. Furthermore, the identification of epitopesin a given protein is readily accomplished using techniques well knownin the art. See, e.g., Geysen et al. (1984) Proc. Natl. Acad. Sci. USA81:3998 (general method of rapidly synthesizing peptides to determinethe location of immunogenic epitopes in a given antigen); U.S. Pat. No.4,708,871 (procedures for identifying and chemically synthesizingepitopes of antigens); and Geysen et al. (1986) Molecular Immunology23:709-715 (technique for identifying peptides with high affinity for agiven antibody). Antibodies that recognize the same epitope can beidentified in a simple immunoassay showing the ability of one antibodyto block the binding of another antibody to a target antigen.

A “unique MenB epitope” is defined herein as an epitope present on aMenB bacterium, wherein antibodies directed toward the epitope arecapable of binding specifically to MenB and not cross reacting, orminimally cross reacting, with sialic acid residues present on thesurface of host tissue. Immunogens containing one or more “unique MenBepitopes” are thus useful in vaccines for the prevention of MenBdisease, and either will not elicit an autoimmune response, or poseminimal risk of eliciting an autoimmune response.

By “mammalian subject” is meant any member of the class Mammalia,including, without limitation, humans and other primates, including suchnon-human primates as chimpanzees and other apes and monkey species;farm animals such as cattle, sheep, pigs, goats and horses; domesticmammals such as dogs and cats; and laboratory animals including rodentssuch as mice, rats and guinea pigs. The term does not denote aparticular age or sex. Thus, both adult and newborn individuals, as wellas fetuses, either male or female, are intended to be covered.

II. Modes of Carrying Out the Invention

As explained above, the native capsular polysaccharide of MenB, termed“MenB PS” herein, is poorly immunogenic in humans and experimentalanimals. Furthermore, native MenB PS elicits the production ofautoantibodies and, hence, may be inappropriate for use in vaccinecompositions. Thus, the present invention uses MenB OS derivativeimmunogens that have been selected based on their ability to elicit theformation of antibodies exhibiting functional activity against MenBbacteria, wherein such functional activity is important in conferringprotection against MenB disease. The immunogens are also selected on thebasis of eliciting an immune response that has minimal or substantiallyundetectable autoimmune activity as determined using the assaysdescribed herein.

More particularly, MenB PS derivatives were prepared for use as startingmaterial in the production of MenB OS derivative immunogens. The MenB PSderivatives generally comprise C₃-C₈ acyl substitutions of sialic acidresidue N-acetyl groups of the native molecule. Particularly preferredMenB PS derivatives comprise the substitution of N-propionyl groups forN-acetyl groups of native MenB PS and are termed “NPr-MenB PS” herein.Such derivatives and methods for synthesizing the same are described ine.g., U.S. Pat. No. 4,727,136 and EP Publication No. 504,202 B, both toJennings et al.

The C₃-C₈ acyl derivatives can be made by first N-deacylating nativeMenB (obtained from e.g., N. meningitidis cultures) in the presence of astrong base to quantitatively remove the N-acetyl groups and to providea reactive amine group in the sialic acid residue parts of the molecule.The deacylated MenB polysaccharides are then N-acylated. For example, inthe case of NPr-MenB PS, the deacylated molecule is N-propionylatedusing a source of propionyl groups such as propionic anhydride orpropionyl chloride, as described in U.S. Pat. No. 4,727,136 to Jenningset al. The extent of N-acylation can be determined using, for example,NMR spectroscopy. In general, reaction conditions are selected such thatthe extent of N-acylation is at least about 80%.

A heterogenous population of high molecular weight MenB PS derivativemolecules is thus obtained. Previous methods have used such heterogenoushigh molecular weight derivatives in glycoconjugate preparations, wherethe derivatives are subjected to controlled periodate oxidation tocreate terminal aldehyde group at the non-reducing end for conjugation(through the aldehydric group at the non-reducing end of thepolysaccharide) to a protein carrier. However, in the practice of thepresent invention, the above-described N-acylated MenB polysaccharidederivatives are fragmented and then size-fractionated to provide asubstantially homogeneous population of intermediate “sized” MenBoligosaccharide fragments for use in preparing glycoconjugates.

In order to provide N-acylated MenB OS derivative-based glycoconjugateshaving well defined and controlled structural configurations,intermediate sized N-acylated MenB oligosaccharides are prepared to havesubstantially homogenous saccharide moiety sizes. Glycoconjugates formedfrom these sized molecules are expected to exhibit more consistentimmunological behavior than heterogenous preparations. Specifically, anN-acylated MenB PS preparation, having substantially 100% N-acylatedsialic acid residues, as determined by, e.g., NMR analysis, can befragmented under mild acidic conditions to provide a population ofoligosaccharide molecules of varying sizes. The fragmented products aresize fractionated, using for example, standard chromatographictechniques combined with e.g., stepwise salt gradients, to providefractions of N-acylated MenB molecules of homogenous sizes. Fractionscontaining intermediate sized oligosaccharides e.g., with an average Dpof about 5 to about 25, preferably 10 to about 20, and more particularlyabout 12 to about 18, are selected for further use herein. These sizedN-acylated oligomers having intermediate length are small enough tofunction as a T-cell-dependent hapten, yet are large enough to expressrelevant conformational epitopes.

In order to increase the immunogenicity of the sized MenB PS derivativefragments, the molecules can be conjugated to a suitable carriermolecule to provide glycoconjugates. Suitable carriers are describedherein further below. Particularly, glycoconjugate preparations havingwell defined and controlled structural configurations can be formed fromintermediate sized N-acylated MenB oligosaccharides to provideN-acylated MenB oligosaccharide immunogens having superiorimmunogenicity.

Thus, in one embodiment of the invention, a group of N-acylated MenB OSglycoconjugates, an example of which is termed “CONJ-1” herein, can beprepared as follows. Fractions of intermediate sized oligosaccharideshaving an average Dp of about 10 to about 20, and preferably about 12 toabout 18, are chemically end-activated at their non-reducing termini andconjugated to protein carriers by a reductive amination technique toprovide the CONJ-1 glycoconjugates. The resulting glycoconjugate isdepicted in FIG. 1, wherein the oligosaccharide fragments 2 are showncovalently linked at their nonreducing ends 4 to a suitable proteincarrier 6 to provide a glycoconjugate, generally indicated at 8.Successful conjugation can be determined using, for example, gelfiltration, and the final saccharide to protein ratio (w/w) assessed bycalorimetric assay.

In a related embodiment of the invention, another group of N-acylatedMenB OS glycoconjugates, an example of which is termed “CONJ-2” herein,can be prepared as follows. Fractions of intermediate sizedoligosaccharides having an average Dp of about 10 to about 20, andpreferably about 12 to about 18, are anchored to a protein carrier attheir reducing ends to provide glycoconjugtes having a reversed chemicalpolarity (orientation). In particular, the reducing ends of theN-acylated MenB oligosaccharide fragments can be converted to free aminogroups by reductive amination using, for example, NaCNBH₃. The freeamino groups can then be modified by covalently attaching an anchoringmolecule bearing an N-OH succinimide active ester of adipic acid.Conjugation to a protein carrier occurs by nucleophilic displacement ofthe active ester group with the ε-amino group of lysine to provide astable amide bond. As can be seen by reference to FIG. 2, the resultingCONJ-2 glycoconjugates, generally indicated at 18, have a configurationsimilar to the CONJ-1 glycoconjugates, however, the saccharide fragmentsare oriented in the opposite direction relative to the protein carrier.This structural orientation more closely resembles the native chemicalpolarity of MenB PS. In particular, the oligosaccharide fragments 12 areshown covalently linked at their reducing ends 15 to a suitable proteincarrier 16 to provide the CONJ-2 glycoconjugate 18.

In order to provide CONJ-2 glycoconjugates wherein the oligosaccharidefragments are projected away from the protein carrier, the above methodcan be altered by using a hydrocarbon spacer arm bearing the N-OHsuccinimide active ester of adipic acid to modify the free amino groupon the aminated molecules. The spacer arm can include a C3-C8 moleculewhich extends the oligosaccharide fragments away from the proteincarrier in the glycoconjugate.

Yet further glycoconjugates can be formed from the above-described sizedMenB OS derivative fragments. In particular, the presence of a lipidmoiety at the reducing ends of bacterial MenB PS has been demonstrated.Mandrell et al. (1982) J. Immunol. 129:2172. Not being bound by anyparticular theory, this lipid moiety may act as an anchoring mechanism,binding the polysaccharide to the bacterial surface through hydrophobicinteractions. Thus, the saccharide-lipid junctional area of native MenBPS may provide unique epitopes (neo-determinants) that are not presentedin purified MenB PS preparations, either as a result of masking and/orshielding effects of the architecture of the long polysialic acid chain,or due to the loss of the lipid moiety during purification.

Accordingly, in still further related embodiments of the invention, MenBOS glycoconjugates are provided, examples of which are termed “CONJ-3”and “CONJ-4” herein, wherein the glycoconjugates are constructed to haveenhanced physicochemical and immunological properties due to theaddition of lipid moieties which provide a mimic of the native MenBsaccharide-lipid junctional area. In one particular embodiment, asubstantially homogeneous fraction of intermediate sizedoligosaccharides having an average Dp of about 15 to about 25 can beobtained as described above. These oligosaccharide fragments should havesufficient length to fold into important conformational epitopes (e.g.,extended helixes), yet are not too long to exert substantial stericmasking or shielding of potential saccharide-lipid junctionneo-epitopes. Hydrocarbon chains of varying length, for example C3-C16long-chain aliphatic lipids, such as phosphatidylethanolamine or otherlipid molecules containing propionyl, hexanoyl and dodecanoyl groups,can be covalently attached at the reducing end of the MenB OS fragmentsusing the above-described N-OH active ester coupling procedure. Theresulting alkylated sialo-oligomers can then be subjected to mildcontrolled periodate oxidation to introduce terminal free aldehydegroups at the nonreducing ends of the oligosaccharide moieties. Thesemonovalent alkylated-sialo-oligomers can then be coupled to a suitableprotein carrier by reductive amination to provide CONJ-3glycoconjugates. Referring to FIG. 3, a CONJ-3 glycoconjugate isgenerally indicated at 28. The glycoconjugate comprises lipid moieties32 covalently attached at the reducing ends 25 of intermediate sizedMenB OS derivative fragments 22 to provide monovalentalkylated-sialo-oligomers, generally indicated at 34. Thealkylated-sialo-oligomers 34 are coupled to a protein carrier 26 at thenonreducing end 24 of the MenB OS fragments 22.

In a related embodiment, a substantially homogeneous fraction ofintermediate sized oligosaccharides having an average Dp of about 15 toabout 25 can be covalently coupled to C3-C16 aliphatic lipids at thenonreducing termini of the MenB OS derivative fragments using periodateoxidation and selective reductive techniques. The resultingalkylated-sialo-oligomers can then be coupled to a suitable proteincarrier by first converting the reducing ends of the MenBoligosaccharide derivative moieties to free amino groups by reductiveamination. The free amino groups can then be modified by covalentlyattaching an anchoring molecule bearing an N—OH succinimide active esterof adipic acid. Optionally, a C3-C8 spacer arm bearing the active estergroup can be added to project the alkylated-sialo-oligomers away fromthe protein carrier. Conjugation to a protein carrier occurs bynucleophilic displacement of the active ester group with the ε-aminogroup of lysine to provide a stable amide bond to provide CONJ-4glycoconjugates. Referring to FIG. 4, a CONJ-4 glycoconjugate isgenerally indicated at 58. The glycoconjugate includes lipid moieties 62covalently attached at the nonreducing ends 54 of intermediate sizedMenB OS derivative fragments 52 to provide monovalentalkylated-sialo-oligomers, generally indicated at 64. Thealkylated-sialo-oligomers 64 are coupled to a protein carrier 56 at thereducing end 55 of the MenB OS fragments 52.

In both of the CONJ-3 and CONJ-4 glycoconjugates, the lipid-saccharidejunctional region is exposed by being arranged distal to the proteincarrier. This configuration renders the lipid-saccharide junctionalregion (neo-epitopes) immunologically accessible and recognizable forinducing antibody formation to the neo-epitope regions. The CONJ-4glycoconjugates have a similar structure to the CONJ-3 glycoconjugates,however, the saccharide fragments are oriented in the opposite directionrelative to the protein carrier.

In addition to providing glycoconjugates such as CONJ-3 and CONJ-4 whichhave artificially generated MenB OS derivatives with lipidated ends,native MenB oligomers which contain the naturally-occurring lipid areisolated and purified for use in preparing glycoconjugate preparations.Thus in another embodiment of the invention, MenB PS can be digestedusing neuraminidase (rather than acid hydrolysis as described above)which preserves the structural and chemical integrity of thesaccharide-lipid portion of the native MenB PS chain. In this manner,sialic acid residues are sequentially removed from the nonreducingterminus by the action of the neuraminidase enzyme. By usingtime-controlled digestion, substantially homogenous fractions ofsialyl-lipid oligomers can be generated having varying chain lengths.The resultant free sialic acid residues can be removed from thepreparation by dialysis, and the retentate, containing the lipid-MenBoligomers can be purified by ion-exchange or hydrophobic interactionchromatography techniques. The lipid-MenB oligomers are then availablefor conjugation to suitable protein carriers using the techniquesdescribed above. In particular, conjugation will generally involveselective end-group activation at the nonreducing end of the lipid-menBoligomers to allow single-site covalent attachment to the carriermolecules.

Each of the above-described glycoconjugates are prepared using carriermolecules that will not themselves induce the production of harmfulantibodies. Suitable carriers are typically large, slowly metabolizedmacromolecules such as proteins, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers, lipidaggregates (such as oil droplets or liposomes), and inactive virusparticles. Preferably, the sized MenB OS derivative fragments of thepresent invention are conjugated to a bacterial toxoid, such as but notlimited to a toxoid from diphtheria, tetanus, cholera, etc. Inparticular embodiments, the oligosaccharide fragments are coupled to theCRM₁₉₇ protein carrier. The CRM₁₉₇ carrier is a well-characterizednon-toxic diphtheria toxin mutant that is useful in glycoconjugatevaccine preparations intended for human use. Bixler et al. (1989) Adv.Exp. Med. Biol. 251:175, Constantino et al. (1992) Vaccine. In otherembodiments, the MenB OS derivative fragments are coupled to proteincarriers known to have potent T-cell epitopes. Exemplary carriersinclude, but are not limited to, Fragment C of tetanus toxin (TT), andthe Class 1 or Class ⅔ OMPs of N. meningitidis. Such carriers are wellknown to those of ordinary skill in the art. Glycoconjugates areselected for their ability to express saccharide-associated epitopesthat mimic those found on the surface of MenB bacterial cells. Suitableglycoconjugates for use with the present invention elicit the formationof functional, bacteria-specific antibodies in immunized hosts, and donot cross-react with host tissue as determined using the binding assaysdescribed herein.

Several factors will have an impact on the physical and immunologicalproperties of the above-described glycoconjugates. Specifically, averageMenB oligomer fragment size, ratio of saccharide to protein (haptenloading density), linkage chemistry, and the choice of protein carrierare all factors that should be considered and optimized in thepreparation of the present glycoconjugates. For example, a lowsaccharide loading density may result in poor anti-saccharide antibodyresponse. On the other hand, a heavy loading of saccharides couldpotentially mask important T-cell epitopes of the protein molecule, thusabrogating the carrier effect and attenuating the total anti-saccharideimmune response.

Accordingly, during the course of the various conjugation reactions,aliquots can be withdrawn and analyzed by SEC-HPLC in order to monitorthe extent of the conjugation process. The use of a disaggregatingbuffer, for example EDTA, SDS, deoxycholate, or the like, can beemployed to separate components possibly adhering to the preparations bynon-covalent interactions. To ensure glycosylation of the carrier, theshift in retention time of the particular protein carrier toward theexclusion volume (V₀) of the column can be monitored. In addition, agradual reduction of the saccharide peak area in a HPLC chromatogram canbe used to indicate incorporation of the saccharide onto the carrier.

Characterization of the glycoconjugates can include molecular weightdetermination using, for example, gel filtration columns. Furthercharacterization may also include electrophoretic mobility on SDS-PAGEseparation equipment and analysis of chemical composition of theglycoconjugates with respect to carbohydrate and amino acid components.The identity of product purity, and the absence of residual contaminants(such as nucleic acids, LPS, and free saccharides and/or carrier) canalso be verified using known techniques. Confirmation of stable covalentattachment can be accomplished using a combination of analyticaltechniques, including gel filtration in detergent-containing buffer,SDS-PAGE followed by Western Blot analysis and amino acid analysis. See,e.g., Vella et al. (1992) Vaccines: New Approaches to InmunologicalProblems, (Ellis, R. W. ed), Butterworth-Heinemann, Boston, pp 1-22,Seid et al. (1989) Glycoconjugate J. 6:489.

The glycoconjugates of the present invention are used to elicit theformation of an anti-MenB immune response in an immunized host.Anti-MenB antibodies produced by the immunized host should bind to MenBbacteria while not cross-reacting, or minimally cross-reacting, withhost tissue sialic acid residues as determined using the binding assaysdescribed herein. The anti-MenB antibodies can be fully characterizedwith respect to isotype, fine antigenic specificity, functional activityand cross-reactivity with polysialic acid residues in host tissue.Glycoconjugates capable of eliciting non-autoreactive, IgG antibodieshaving bactericidal activity are selected for use in preparing vaccineformulations for use in anti-MenB immunization.

For example, immunogenicity of MenB OS derivative glycoconjugates can bedetermined by challenging mammalian subjects, conveniently, standardlaboratory animals such as rodents and rabbits, with compositionscontaining the glycoconjugates along with a suitable adjuvant, describedfurther below. Groups of subjects are generally immunized and boostedseveral times with the compositions, or with control materials (e.g.,adjuvant alone, native MenB PS, MenB OS derivative fragments, ornon-covalent MenB OS derivative/carrier complexes). Antisera fromimmunized subjects can be obtained, and serial dilutions of pooled seraevaluated by, e.g., ELISA using standard techniques. Labeled anti-IgGsera can be used to measure IgG anti-MenB OS derivative antibodyresponse. In order to determine the isotypes of the antibodies elicitedby the conjugates, standard methods, such as ELISAs, can also be runusing labelled molecules specific for IgG subclasses IgG1, IgG2a, IgG2band IgG3. An isotypic response that is predominantly IgG1 along withIgG2b and, to a lesser extent, IgG2a and IgG3 is characteristic of aT-cell dependent antigen. Conjugates that are found to be highlyimmunogenic and produce predominantly IgG antibodies are selected forfurther evaluation.

In particular, the specificity of the antibodies elicited by selectedMenB OS derivative glycoconjugates can be further evaluated usingcompetitive specific binding assays, such as inhibition ELISA, or thelike. For example, antisera obtained from immunized subjects, along witheither soluble MenB OS derivatives (or glycoconjugates) or native MenBPS, can be reacted with bound MenB OS derivatives (or glycoconjugatesthereof) in a suitable ELISA reaction vessel using labeled anti Ig(anti-IgM, IgG and IgA) as the secondary antibody. MenB OSglycoconjugates that elicit the formation of antibodies that areinhibited to a greater extent by the soluble MenB OS derivatives andglycoconjugates than by the soluble native MenB PS (e.g., that elicitantibodies which exhibit a higher affinity for the modifiedpolysaccharide molecule) are thus selected as candidates for use infurther immunization studies.

Functional activity can be determined by assessing complement-mediatedbactericidal activity and/or opsonic activity. In particular,complement-mediated bactericidal activity of the antibodies can beevaluated using standard assays such as those described by Gold et al.(1970) Infect. Immun. 1:479, Westerink et al. (1988) Infect. Immun.56:1120, Mandrell et al. (1995) J. Infect. Dis. 172:1279, and Granoff etal. (1995) Clin. Diagn. Laboratory Immunol. 2:574. In these assays, N.meningitidis is reacted with a complement source as well as with theantibody to be tested. Bacterial counts are done at various samplingtimes. Those antibodies that demonstrate complement-mediatedbactericidal activity, as demonstrated by a minimum of a 50% reductionin viable bacterial cell counts determined after sixty minutesincubation with antibody and complement, as compared to colony counts attime zero, are considered to exhibit bactericidal activity for purposesof the present invention and are suitable for further use.

Complement-mediated bacteriolysis is thought to be the major mechanismresponsible for host protection against invasive Meningococcal disease.However, considerable evidence also supports an important protectiverole for opsonization (see, e.g., Bjerknes et al. (1995) Infect. Immun.63:160). Accordingly, the opsonic activity of the antibodies producedherein can be evaluated as a second measure, or as an alternativemeasure, to assess functional activity. Results from opsonic assays canbe used to supplement bactericidal data, and to help in the selection ofappropriate glycoconjugates capable of conferring protection.

A variety of opsonic assay methods are known in the art, and can be usedto evaluate functional activity of antibodies induced by theglycoconjugates of the present invention. Such standard assays includethose described by Sjursen et al. (1987) Acta Path. Microbiol. Immunol.Scand., Sec. C 95:283, Halstensen et al. (1989) Scand. J. Infect. Dis.21:267, Lehmann et al. (1991) APMIS 99:769, Halstensen et al. (1991)NIPH Annals 14:157, Fredlund et al. (1992) APMIS 100:449, Guttormsen etal. (1992) Infect. Immun. 60:2777, Guttormsen et al. (1993) J. Infec.Dis. 167:1314, Bjerknes et al. (1995) Infect. Immun. 63:160, Hayrinen etal. (1995) J. Infect. Dis. 171:1481, de Velasco et al. (1995) J. Infect.Dis. 172:262, and Verheul, A. F. M. (1991) “Meningococcal LPS DerivedOligosaccharide-Protein Conjugate Vaccines, Immunochemical andImmunological Aspects,” Thesis, Utrecht University, The Netherlands, pp.112-135.

Several binding assays can be used to evaluate possible autoreactivityof antibodies induced by the glycoconjugates of the present invention.In particular, the induced antibodies can be evaluated for their abilityto bind to host cells which express polysialic acid residues on theircell surfaces. Such cells represent surrogate targets for the detectionof antibodies that exhibit autoimmune activity. One target comprises thehuman neuroblastoma cell line, CHP-134, which expresses long chain α2-8polysialic acid (NCAM) on its cell surface, as described by Livingstonet al. (1988) J. Biol. Chem. 263:9443. Other suitable targets include,but are not limited to, newborn brain cells, tissues derived from e.g.,kidney, heart and the olfactory nerve, cultured saphenous veinendothelial cells, cytotoxic T lymphocytes and natural killer (NK)cells. See, e.g., Brandon et al. (1993) Intl. J. Immunopathology andPharmacology 6:77. Antibody molecules obtained from immunized subjectscan be added to suitable test cell populations in culture, and thepotential binding of the antibodies to the cellular targets detected andquantified directly using labeled monoclonals, or indirectly using anappropriately labeled secondary reagent that reacts specifically withthe antibody (e.g., Staphylococcal Protein A and G and anti-murineantibody molecules). Antibodies that do not cross-react with test hosttissue PSA or that display minimal reactivity are not consideredautoreactive for purposes of the present invention. Thus, theglycoconjugates used to elicit formation of such antibodies areappropriate for further use. In addition, some antibodies that showbinding with test tissue, which binding is not affected by pre-treatmentof the test cells with neuraminidase, may also be indicative ofglycoconjugates that are appropriate for further use. Autoreactivity ofsuch antibodies is termed “indeterminate” herein.

The processes used to provide the various MenB OS-derivative conjugatesare designed to produce superior immunogens presenting uniquesaccharide-associated epitopes that mimic those found on the surface ofMenB organisms and are expressed minimally in the host. The saccharidederivatives described herein are thus capable of eliciting theproduction of MenB-specific antibodies, and are used directly inanti-MenB vaccine formulations which can be used in pharmaceuticalcompositions to prevent and/or treat MenB and E. coli K1 disease inmammals. Such disease includes bacterial meningitis and sepsis ininfants, children and adults.

The vaccines can comprise one or more of the MenB OS derivativeimmunogens. The vaccines may also be administered in conjunction withother antigens and immunoregulatory agents, for example,immunoglobulins, cytokines, lymphokines, and chemokines, including butnot limited to IL-2, modified IL-2 (cys125→ser125), GM-CSF, IL-12,γ-interferon, IP-10, MIP1β and RANTES.

The vaccines will generally include one or more “pharmaceuticallyacceptable excipients or vehicles” such as water, saline, glycerol,ethanol, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, and the like, may bepresent in such vehicles.

Adjuvants may also be used to enhance the effectiveness of the vaccines.Adjuvants can be added directly to the vaccine compositions or can beadministered separately, either concurrent with or shortly after,vaccine administration. Such adjuvants include, but are not limited to:(1) aluminum salts (alum), such as aluminum hydroxide, aluminumphosphate, aluminum sulfate, etc.; (2) oil-in-water emulsionformulations (with or without other specific immunostimulating agentssuch as muramyl peptides (see below) or bacterial cell wall components),such as for example (a) MF59 (International Publication No. WO90/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85(optionally containing various amounts of MTP-PE (see below), althoughnot required) formulated into submicron particles using a microfluidizersuch as Model 110Y microfluidizer (Microfluidics, Newton, Mass.), (b)SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymerL121, and thr-MDP (see below) either microfluidized into a submicronemulsion or vortexed to generate a larger particle size emulsion, and(c) Ribi™ adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.)containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cellwall components from the group consisting of monophosphorylipid A (MPL),trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferablyMPL+CWS (Detox™); (3) saponin adjuvants, such as Stimulon™ (CambridgeBioscience, Worcester, Mass.) may be used or particle generatedtherefrom such as ISCOMs (immunostimulating complexes); (4) CompleteFreunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5)cytokines, such as interleukins (IL-1, IL-2, etc.), macrophage colonystimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; and (6)other substances that act as immunostimulating agents to enhance theeffectiveness of the composition.

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP),N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

Typically, the vaccine compositions are prepared as injectables, eitheras liquid solutions or suspensions; solid forms suitable for solutionin, or suspension in, liquid vehicles prior to injection may also beprepared. The preparation also may be emulsified or encapsulated inliposomes for enhanced adjuvant effect, as discussed above.

The vaccines will comprise a therapeutically effective amount of theMenB OS derivative glycoconjugate immunogen, and any other of theabove-mentioned components, as needed. By “therapeutically effectiveamount” is meant an amount of a molecule which will induce animmunological response in the individual to which it is administeredwithout stimulating an autoimmune response. Such a response willgenerally result in the development in the subject of a secretory,cellular and/or antibody-mediated immune response to the vaccine.Usually, such a response includes but is not limited to one or more ofthe following effects; the production of antibodies from any of theimmunological classes, such as immunoglobulins A, D, E, G or M; theproliferation of B and T lymphocytes; the provision of activation,growth and differentiation signals to immunological cells; expansion ofhelper T cell, suppressor T cell, and/or cytotoxic T cell and/or γδ Tcell populations.

Preferably, the effective amount is sufficient to bring about treatment,i.e., reduction or complete elimination of symptoms, or prevention ofdisease symptoms. The exact amount necessary will vary depending on thesubject being treated; the age and general condition of the subject tobe treated; the capacity of the subject's immune system to synthesizeantibodies; the degree of protection desired; the severity of thecondition being treated; the particular molecule selected and its modeof administration, among other factors. An appropriate effective amountcan be readily determined by one of skill in the art. A “therapeuticallyeffective amount” will fall in a relatively broad range that can bedetermined through routine trials.

Once formulated, the vaccines are conventionally administeredparenterally, e.g., by injection, either subcutaneously orintramuscularly. Additional formulations suitable for other modes ofadministration include oral and pulmonary formulations, suppositories,and transdermal applications. Dosage treatment may be a single doseschedule or a multiple dose schedule.

III. Experimental

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

EXAMPLE 1 Preparation of Control MenB PS Conjugates

Purified MenB PS in its sodium form was deacylated using 2M NaOH andNaBH₄ at about 110° C. for 6 hours to quantitatively remove the N-acetylgroups. The deacylated MenB PS was N-propionylated by use of propionicanhydride to yield NPr-MenB PS, as described in U.S. Pat. No. 4,727,136to Jennings et al. The extent of N-propionylation was estimated to bearound 84% by ¹H-NMR spectroscopy. The NPr-MenB PS was purified bydialyzing against distilled water, and subjected to mild periodateoxidation to introduce a terminal aldehydric group at the non-reducingend for subsequent conjugation to a protein carrier, as described inU.S. Pat. No. 4,727,136 to Jennings et al. During the mild periodateoxidation, the NPr-MenB PS was fragmented, giving rise to a heterogenouspopulation of “unsized” NPr-MenB oligosaccharide fragments, typicallyhaving an average DP of greater than about 30.

Conjugation of the unsized NPr-MenB PS fragments to two differentprotein carriers, TT and CRM₁₉₇, was performed by reductive amination inthe presence of sodium cyanoborohydride. The conjugation reaction wascarried out over about 5 days at 40° C. Two control MenB PS conjugateswere thus obtained, C1/TT and C1/CRM₁₉₇.

EXAMPLE 2 Characterization of the Control Conjugates

SDS-PAGE and SEPHADEX G-100 gel filtration were carried out in order toconfirm formation of covalent conjugate moieties. Referring to FIGS. 5Aand 5B, the results of a typical SEPHADEX G-100 gel filtration of theC1/CRM₁₉₇ conjugate is depicted. In particular, the chromatogram of anoncovalent mixture of the NPr-MenB PS fragments and the CRM₁₉₇ carriermolecule prior to conjugation, (e.g., before the addition of NaCNBH₃) isdepicted in FIG. 5A. As can be seen, the NPr-MenB saccharides eluted asa broad peak near the bed volume, while the CRM₁₉₇ protein elutedslightly ahead of bed volume. The chromatogram after conjugation (e.g.,following the addition of NaCNBH₃ to effect reductive amination) isdepicted in FIG. 5B. As shown therein, a new high molecular weight (HMW)peak appeared near the void volume. This HMW peak, containing bothsaccharide and protein, was collected and identified as the C1/CRM₁₉₇conjugate.

The final saccharide-to-protein ratios (w/w) of the two C1conjugateswere determined by calorimetric assays and found to be 0.21 for theC1/CRM₁₉₇ conjugate and 0.15 for C1/TT conjugate.

EXAMPLE 3 Immunogenicity of the Control Conjugates

In order to assess the ability of the C1 control conjugates to elicit anIgG anti-NPr-MenB PS antibody response in an immunized subject, thefollowing study was carried out. Groups of CD1 mice (10 animals/group)were immunized three times by intraperitoneal (ip) injection usingvaccine formulations containing C1/TT or C1/CRM₁₉₇ conjugates with FCAor alum adjuvant (5.0 μg sialic acid content in the conjugate vaccinefor the first injection, and 2.5 μg sialic acid content in the secondand third doses). Control groups were immunized with either adjuvantalone; native MenB PS; NPr-MenB PS; or noncovalently associated NPr-MenBPS/carrier complexes.

Serum samples were collected and pooled from each experimental groupconcurrently with each immunization boost, as well as 11 days after thefinal boost. Serial dilutions of pooled sera were made and evaluated byELISA using an avidin-biotinylated NPr-MenB PS system. After overnightincubation with the sera, the reaction wells were incubated for 3 hourswith alkaline phosphatase-labelled anti-murine sera specific to IgG.After washing, p-nitrophenyl phosphate was added to the wells, and theoptical density (“OD”) values were read at 405 nm after 30 minutes colordevelopment. The OD values are reported in FIG. 6. Both the C1/TT andC1/CRM₁₉₇ conjugates were immunogenic when administered with FCA, andimmunogenic to a lesser extent when administered with the alum adjuvant.The OD values of the pooled sera from animals immunized with theconjugate/FCA vaccine formulations are shown at 1:1600 sera dilution,while the OD values of the pooled sera from animals immunized with theconjugate/alum vaccine formulations are shown at 1:400 sera dilution. Ascan be seen by reference to FIG. 6, there was no significant differenceobserved in immunogenicity due to the particular protein carrier used(TT or CRM₁₉₇); however, use of the FCA adjuvant greatly increased theimmunogenicity of the vaccine compositions.

EXAMPLE 4 Characterization of the Antibody Response Elicited by the C1Conjugates

In order to evaluate the IgG subclass of the antibody response inducedby the C1 control conjugate, the following study was carried out. Groupsof mice (10 animals per group) were given three doses of vaccinecompositions containing either C1/TT or C1/CRM₁₉₇ conjugate with FCA oralum adjuvant. The dosages were the same as those used in theimmunizations of Example 3 above. Serum samples were collected andpooled from each experimental group after the final immunization boost.Serial dilutions of pooled sera were made and evaluated by ELISA usingan avidin-biotinylated NPr-MenB PS system. After overnight incubation ofsera, reaction wells were incubated for 3 hours with alkalinephosphatase-labelled anti-murine sera specific to IgG subclasses IgG1,IgG2a, IgG2b and IgG3. After washing, p-nitrophenyl phosphate was addedto the wells, and the OD values were read at 405 nm after 30 minutescolor development. The OD values are depicted in FIG. 7, wherein thevalues represent the net OD after subtraction of blank values obtainedfrom wells containing only the calorimetric substrate.

As can be seen, the predominant antibody response was IgG1; however,when the conjugates were administered with the FCA adjuvant, there alsowere IgG2b and, to a lesser extent, IgG2a and IgG3 antibody responses.Thus, the antibody response elicited in the immunized mice by C1conjugates is characteristic of a T-cell dependent antigen.

In order to evaluate total anti-Npr-MenB PS antibody response induced bythe C1/TT and C1/CRM₁₉₇ conjugates in CD1 mice, and to determine thespecificity of the conjugate-induced antibody responses, the followingstudy was carried out. Groups of CD1 mice (8 to 10 animals per group)were immunized with three doses of conjugate vaccine formulations orcontrol materials as described above in Example 3. Serum samples werecollected and pooled from each experimental group after the finalimmunization boost. In order to assess total Ig response to the C1conjugates, solid phase ELISA was carried out wherein biotinylatedNPr-MenB PS (bound to the reaction wells by avidin) was used as thecoating antigen. The labelling antibody was anti-murine IgM, IgG and IgAconjugated to alkaline phosphatase. After washing, p-nitrophenylphosphate was added to the wells, and the OD values were read at 405 nmafter 30 minutes color development. In order to assess specificity,competitive inhibition ELISA was carried out using the same coatingantigen with the addition of either soluble NPr-MenB PS or native MenBPS inhibitors at 25 μg/ml.

The results of both the determination of the level of total antibodyresponse and the specificity of the responding antibodies are depictedbelow in Table 1.

TABLE 1 Mouse 1/Titer % Inhibition Group Vaccine Adjuvant (OD 0.5) NPrForm NAc Form 1 C1 (TT) FCA ˜7,000 97 28 conjugate 2 C1 (TT) alum ˜1,20099 14 conjugate 3 none FCA <100 — — 4 none alum <100 — — 5 NPr-MenB FCA<100 — — OS + TT 6 NPr-MenB PS FCA <100 — 7 NPr-MenB PS none <100 — 8MenB PS none <100 — — 9 C1 (CRM₁₉₇) FCA ˜6,000 98 36 conjugate 10 C1(CRM₁₉₇) alum ˜175 56  ˜0 conjugate 11 NPr-MenB alum <100 — — OS +CRM₁₉₇

As can be seen in Table 1, the CD1 mice that were immunized with the C1conjugate/FCA adjuvant formulations gave significant antibody responsesto NPr-MenB PS. In addition, the antibody response was specific toNPr-MenB PS as demonstrated by almost complete inhibition (e.g., 97-99%)by the soluble NPr-MenB inhibitor as compared with the partialinhibition (e.g., 14-36%) observed with the native MenB PS inhibitor.The percent inhibition with the soluble inhibitors is expressed in Table1 as a comparison with buffer controls.

In order to assess bactericidal activity, pooled sera obtained from theabove immunized subjects was added to cultures of N. meningitidis (MenBbacteria cultures) along with a source of complement. In this particularassay, a heterologous complement source (e.g., juvenile rabbit serum)was used. Negative (sera) control and complement control cultures werealso assayed, and all sera were heat-inactivated before testing. Theresults of the bactericidal assay are depicted below in Table 2.

TABLE 2 Mice Group Vaccine 1/BC₅₀ ¹ 1 C1 (TT)/FCA ˜50 9 C1 (CRM₁₉₇)/FCA˜200 2 C1 (TT)/alum <25 10  C1 (CRM₁₉₇)/alum <25 3,4,5,6 ControlGroups^(2,3) <25 ¹The BC₅₀ is the reciprocal of the dilution of thepost-3rd pooled sera at which 50% of the bacteria were killed, relativeto the negative sera controls and complement controls. ²Negative controlGroups: Group 3: FCA Group 4: Alum Group 5: NPr-MenB PS + TT(noncovalent) Group 6: NPr-MenB PS + FCA ³ Complement controls:Anti-MenY MAb + C′ = negative Anti-MenB porin MAb + C′ = positiveAnti-MenB porin MAb − C′ = negative Mouse group sera − C′ = negative

In Table 2, bactericidal activity is expressed as the concentration atwhich 50% of the MenB bacteria were killed relative to the negative seracontrols and complement controls. As can be seen, CD1 mice immunizedwith the C1/TT and C1/CRM₁₉₇ conjugates administered with FCA producedantibodies that demonstrate significant bactericidal activity.

EXAMPLE 5 Preparation of CONJ-1 MenB OS Derivative Glycoconjugates

A preparation of NPr-MenB OS derivative-tetanus toxoid conjugates,hereinafter referred to as CONJ-1, was prepared as follows. PurifiedMenB PS in its sodium form was deacylated with 2M NaOH at about 110° C.for 6 hours to quantitatively remove the N-acetyl groups. The alkalitreatment was performed in the presence of NaBH₄. After alkalitreatment, the deacylated MenB PS was exhaustively dialyzed in saturatedsodium bicarbonate buffer. The dialyzed product was then treated with anexcess of propionic anhydride with stirring overnight at ambienttemperature to yield NPr-MenB PS. The NPr-MenB PS was exhaustivelydialyzed in water and recovered by lyophilization. The extent ofN-propionylation as measured by ¹H-NMR spectroscopy was found to besubstantially 100%.

The NPr-MenB PS was depolymerized (fragmented) under mild acidicconditions (e.g., 10 mM acetate, pH 5.5 at 50° C. for 2 hours) to give amixture of NPr-MenB oligosaccharides (NPr-MenB OS) of varying sizes. Thekinetics of hydrolysis of the NPr-MenB PS, and the resulting fragmentedoligosaccharide profile can be monitored by analytical FPLC monoQchromatography.

The mixture of fragmented NPr-MenB OS was size-fractionated onQ-Sepharose with a low (100 mM NaCl) and high (500 mM NaCl) stepwisesalt gradient. By analytical analysis, for example, the Svennerholmresorcinol assay for sialic acid (Svennerholm, L. (1957) Biochim.Biophys. Acta 24:604) and the Hantzsch calorimetric assay for releasedformaldehyde from the non-reducing end of NPr-MenB PS oligomers (Nash,T. (1953) Biochem. J. 55:416), the 100 mM NaCl fraction should containsmall-sized NPr-MenB OS molecules with an average Dp of 3-6 and the 500mM NaCl fraction should contain intermediate-sized NPr-MenB OS moleculeswith an average Dp of 13-20. As shown by analytical monoQ analysis overa Q-Sepharose column, the expected oligosaccharide distribution patternwas confirmed wherein the 100 mM NaCl fraction contained small oligomers(i.e., average Dp of 2.85) and the 500 mM NaCl fraction containedintermediate size oligomers with an average Dp of 13.

A group of intermediate-sized NPr-MenB OS derivatives (Dp of 13)recovered from the 500 mM NaCl fraction of Q-Sepharose were chemicallyend-activated at their non-reducing termini and conjugated to tetanustoxoid (TT) by a reductive amination method to provide CONJ-1glycoconjugates. More particularly, the Dp 13 oligosaccharides weresubjected to mild periodate oxidation (e.g., 100 mM sodium perborate for15-30 minutes in the dark at ambient temperature) to introduce aterminal aldehydric group at the non-reducing ends of theoligosaccharides. Following periodate oxidation, excess ethylene glycolwas used to quench the oxidation reaction. The oxidized,intermediate-sized NPr-MenB oligosaccharide derivatives were purified bydesalting on a SEPHADEX G-25 column and then lyophilized.

The reductive amination conjugation reaction was performed in thepresence of sodium cyanoborohydride for 3 to 5 days. For the conjugationreaction, the saccharide-to-protein ratio can range from 50 to 250mol/mol. To prepare the NPr-MenB OS/TT conjugates, the pool of NPr-MenBoligomers with average Dp13 was combined with suitably prepared TT at aninitial high molar ratio (200:1) of oligomer-to-protein. The reactionwas allowed to proceed for 3 days (e.g., 1 day at 40° C., followed by 2days at ambient temperature).

Isolation and purification of the CONJ-1 glycoconjugates can beaccomplished by gel permeation chromatography with an appropriate sizingcolumn or by hydrophobic interaction chromatography (e.g., using PhenylSepharose). Either chromatographic procedure is efficient in separatingthe glycoconjugates from reagents, byproducts, and unreacted saccharideand protein carrier molecules.

EXAMPLE 6 Characterization of the NPr-MenB OS Derivative CONJ-1Glycoconjugate

The CONJ-1 glycoconjugate was characterized as follows. In order todemonstrate covalence (e.g., establishing a covalent linkage between theNPr-MenB OS and the protein carrier), a number of physico-chemicaltechniques can be used, including: SDS-PAGE; Western Blot; SEPHADEXG-100 gel filtration; amino acid analysis; or the like. For the purposesof the present study, SDS-PAGE was used to establish covalent attachmentof the NPR-MenB OS/TT CONJ-1 glycoconjugates by revealing a shift tohigher molecular weight for the conjugate band as compared to thecarrier protein band, per se. Western blot analysis of the CONJ-1glycoconjugates demonstrated covalence by the coincidence of positivesignals for TT and NPr-MenB OS with specific anti-TT and anti-NPr-MenBOS antisera.

Based on steric factors, the use of oligosaccharides instead of largemolecular weight polysaccharides in the preparation of the CONJ-1glycoconjugates allows for higher coupling efficiency of saccharideantigens onto the protein carrier molecule. The finalsaccharide-to-protein ratio of these NPr-MenB oligosaccharide-basedconjugates range from about 0.10 to 0.25 which corresponds to about 3 to5 NPr-MenB oligosaccharide chains covalently bound per protein carrier.On a per weight basis, the CONJ-1 glycoconjugates appear to have ahigher saccharide loading than a previously reported NPr-MenB PS-basedconjugate (U.S. Pat. No. 4,727,136) which contains, on the average,about 7.5 to 18.8 times more saccharide (using 10,000 Daltons as themolecular weight of NPr-MenB PS)

In addition, constructing the CONJ-1 glycoconjugates to havesubstantially homogenous-sized saccharide moieties of an intermediatechain length (e.g., average Dp of 10-20) is expected to result inglycoconjugates which display more consistent immunological behavior.Further, the selective end-activation (e.g., selective introduction ofthe aldehyde group at the non-reducing terminus) of the Q-Sepharosechromatography-purified NPr-MenB oligosaccharides avoids the possibilityof cross-linked, heterogenous structures which could arise from the useof NPr-MenB PS molecules with “active” aldehyde groups introduced atboth termini. In this regard, it is likely that bi-terminally activatedpolysaccharide molecules (having aldehyde groups at both ends) could bederived from a periodate oxidation of N-acylated MenB PS previouslyexposed to NaBH₄ during the N-deacetylation procedure.

EXAMPLE 7 Evaluation of Immunogenicity of the NPr-MenB OS DerivativeGlycoconjugates

Groups of 4 to 6 week old CD1 and BALB/c mice, 5 to 6 animals per group,were vaccinated with 3 doses of a vaccine composition formed fromNPr-MenB OS/TT (CONJ-1) glycoconjugate and FCA adjuvant. A negativecontrol group was vaccinated with the FCA adjuvant alone. Vaccinationsand boosts were administered 3.5 to 4 weeks apart. Pooled sera,collected after the second boost, were analyzed for ELISA titers toNPr-MenB OS. As a positive control, pooled sera from a second post-boostimmunization of CD1 mice vaccinated with a second conjugate, NPr-MenBOS-CRM₁₉₇ (termed CONJ-1/CRM₁₉₇) was used. Antibody specificity was alsodetermined using soluble NPr-MenB OS inhibitor (25 μg/mL) in acompetitive inhibition ELISA. Inhibition by soluble native MenB PS(NAc-MenB PS) (25 μg/mL) was also measured. For the ELISAS, biotinylatedNPr-MenB PS (bound to avidin coated plates) was used as the coatingantigen. The labelling antibody was anti-murine Ig (anti-IgM, IgG andIgA) conjugated to alkaline phosphatase. The results of the ELISAs aredepicted below in Table 3.

TABLE 3 % Inhibition¹ MenB Mice Pooled 1/Titer Polysaccharides GroupSera Vaccine Adjuvant (OD 1.0) NPr NAc CD1 CONJ-1 FCA ˜12,800 90 12 (TT)conjugate BALB/c CONJ-1 FCA ˜3,200 89  4 (TT) conjugate NegativeNPr-MenB FCA <100 — — control sera OS FCA (CRM₁₉₇) Conjugate PositiveNPr-MenB FCA ˜1,600 87 11 control sera* OS (CRM₁₉₇) Conjugate ¹Percentinhibition with the soluble inhibitors shown (at 25 μg/ml) compared withbuffer controls. *See text.

The data depicted in Table 3 represent titers obtained using the net ODafter subtraction of OD values in wells containing serum diluted withsoluble NPr-MenB PS so as to report only inhibitable binding. Thisprocedure avoids reporting of nonspecific binding in the assay (see,e.g., Granoff et al. (1995) Clinic. Diag. Lab. Immunol. 2:574). Percentinhibition with the soluble molecules also is reported as compared withbuffer controls. Antibody response elicited by the CONJ-1glycoconjugates was specific for NPr-MenB saccharide derivatives asevidenced by 80-90% competitive inhibition by the soluble NPr-MenB PS.In contrast, only 4-12% inhibition was observed when soluble NAc-MenB PSwas used. As can be seen, both CD1 and BALB/c mice gave significantantibody responses to NPr-MenB PS when immunized with the CONJ-1glycoconjugates as compared with the negative controls (immunizationswith FCA only). ELISA titers in CD1 mice were higher than those obtainedwith the BALB/c mice.

Thus, novel MenB OS derivative-immunogens, and methods for obtaining andusing the same are disclosed. Although preferred embodiments of thesubject invention have been described in some detail, it is understoodthat obvious variations can be made without departing from the spiritand the scope of the invention as defined by the appended claims.

What is claimed is:
 1. A glycoconjugate comprising substantiallyhomogenous sized Neisseria meningitidis serogroup B (MenB) capsularoligosaccharides in which sialic acid residue N-acetyl groups arereplaced with saturated N-propionyl groups, wherein said MenBoligosaccharides are covalently attached to a protein carrier molecule,have an average degree of polymerization (Dp) of 10 to 20, and furthercomprise a C3-C16 long-chain aliphatic lipid covalently attachedthereto; and wherein said glycoconjugate has a saccharide-to-proteinratio ranging from 0.10 to 0.25 by weight, and elicits antibodies thatdo not bind to the human neuroblastoma cell line CHP-134.
 2. Theglycoconjugate of claim 1 wherein the carrier molecule is a bacterialtoxoid.
 3. The glycoconjugate of claim 2 wherein the bacterial toxoid istetanus toxoid.
 4. The glycoconjugate of claim 1 wherein the carriermolecule is a nontoxic mutant bacterial toxoid.
 5. The glycoconjugate ofclaim 4 wherein the mutant bacterial toxoid is CRM₁₉₇.
 6. Theglycoconjugate of claim 1 wherein the MenB OS derivatives have anaverage Dp of about 12 to about
 18. 7. A glyconjugate comprisingsubstantially homogenous sized Neisseria meningitidis serogroup B (MenB)capsular oligosaccharides in which sialic acid residue N-acetyl arereplaced with saturated N-propionyl groups, wherein said MenBoligosaccharides are covalently attached to a CRM₁₉₇ toxoid proteincarrier, have an average Dp of 12 to 18, and further comprise a C3-C16long-chain aliphatic lipid covalently attached thereto, and wherein saidglycoconjugate has a saccharide-to-protein ratio ranging from 0.10 to0.25 by weight, and elicits antibodies that do not bind to the humanneuroblastoma cell line CHP-134.
 8. A vaccine composition comprising thecombination of: a glycoconjugate formed from substantially homogenoussized Neisseria meningitidis serogroup B (MenB) capsularoligosaccharides in which sialic acid residue N-acetyl groups arereplaced with saturated N-propionyl groups wherein said MenBoligosaccharides are covalently attached to a protein carrier molecule,have an average degree of polymerization (Dp) of 10 to 20, and furthercomprise a C3-C16 long-chain aliphatic lipid covalently attachedthereto, and wherein said glycoconjugate has a saccharide-to-proteinratio ranging from 0.10 to 0.25 by weight and, elicits antibodies thatdo not bind to the human neuroblastoma cell line CHP-134; and apharmaceutically acceptable excipient.
 9. The vaccine composition ofclaim 8 wherein the MenB oligosaccharides have an average Dp of about 12to about
 18. 10. A vaccine composition comprising the combination of: aglycoconjugate formed from substantially homogenous sized Neisseriameningitidis serogroup B (MenB) capsular oligosaccharides in whichsialic acid residue N-acetyl groups are replaced with saturatedN-propionyl groups, wherein said MenB oligosaccharides are covalentlyattached to a CRM₁₉₇ toxoid protein carrier and have an average degreeof polymerization (Dp) of 12 to 18, and the MenB OS derivatives furthercomprise a C3-C16 long-chain aliphatic lipid covalently attached theretoand wherein said glycoconjugate has a saccharide-to-protein ratioranging from 0.10 to 0.25 by weight and, elicits antibodies that do notbind to the human neuroblastoma cell line CHP-134; and apharmaceutically acceptable excipient.
 11. The vaccine composition ofclaim 8 further comprising an adjuvant.
 12. The vaccine composition ofclaim 10 further comprising an adjuvant.